This project is designed to provide information on basic neural mechanisms involved in the generation and control of respiratory movements in mammals. The long-range goal is to explain the ontogeny and neurogenesis of respiratory movements in terms of the molecular, biophysical, synaptic, and network properties of respiratory neurons in the mammalian brainstem and spinal cord. Current work focuses on cellular and network mechanisms generating the respiratory rhythm in the brainstem. A set of interrelated, multidisciplinary studies are ongoing to determine: (1) sites, cellular components, and architecture of brainstem networks involved in generation and transmission of respiratory rhythm; (2) biophysical properties and synaptic interactions of neurons forming the respiratory oscillator; (3) neurochemical mechanisms for modulation and synaptic transmission of rhythm; and (4) molecular properties of functionally identified neurons. Experiments are performed with isolated in situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal and juvenile rodents. Previously we have identified the brainstem locus (called the pre-Botzinger complex) containing populations of neurons participating in rhythm generation. We have further developed novel methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing infrared and differential interference contrast (IR-DIC) imaging performed simultaneously with fluorescence imaging of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of respiratory network neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of neuron channel and receptor expression. With these approaches, we have imaged the activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex in vitro, providing the most direct experimental evidence to date that rhythm generation involves a network of neurons with specialized cellular properties. Methods for multi-photon imaging that will allow three-dimensional reconstruction of this network in the pre-Botzinger complex are currently under development. Studies of cellular membrane biophysical properties have provided additional evidence that persistent sodium and potassium leak conductances represent critical ionic conductance mechanisms for rhythm generation. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons shows a profile of sodium and potassium channels consistent with an important role of persistent sodium and potassium leak conductances. Electrophysiological studies have also demonstrated that these conductance mechanisms are critically involved in the regulation of the breathing rhythm by a diverse set of neurochemicals that modulate these conductances, including serotonin and substance P, as well as physiological control signals including carbon dioxide and oxygen. Electrophysiological studies performed with more intact preparations of the brainstem-spinal cord in situ have confirmed the importance of these cellular and network mechanisms in both the neonatal and mature mammalian nervous systems. These results continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of the breathing rhythm in the intact mammalian nervous system. Novel computational approaches including large-scale modeling of brainstem neural networks have been used in parallel to experimental studies. Our biophysically realistic computational models of respiratory neurons have been further developed and novel investigations were conducted on the dynamic behavior of synaptically coupled populations of these network cells. Computer simulations with these models mimic many features of the single-cell and neuron population activity found experimentally in vitro and in situ, including instabilities of the rhythm produced by nonlinear dynamic phenomena such as quasiperiodicity arising in networks. These models are currently being applied to further explore principles of operation of brainstem respiratory networks at different stages of nervous system development.